Method of identification and quantification of proteins, isoforms of the angiotensin i converting enzyme

ABSTRACT

The present invention relates to a method of identification and quantification of proteins, isoforms of angiosttensin I converting enzymes (ACE), 190-kDa, specially of 90 kDa in tissues, cells and biological fluids, specially in urine, genetic marker and prognostic agent of hypertension and primary or secondary renal lesion and kit for using in the diagnosis, risk stratification and threapeutical decision in arterial hypertension. One the aims of the present invention is to check the potential of the 90 kDa isoform as a hypertension genetic marker isoform and as a prognostic for hypertension.

FIELD OF INVENTION

The present invention relates to a method of identification andquantification of proteins, isoforms of angiotensin I converting enzyme(ACE), specifically ACE of 190-kDa, specially of 90 kDa (genetic markerof hypertension) and of 65 kDa in tissues, cells and biological fluids,specially in urine, a molecular marker based on said proteins, use ofmentioned molecular marker, analytical method for diagnosis, riskstratification, therapeutical decision in carriers of arterialhypertension and primary or secondary renal lesion and kit for using inthe diagnosis.

BACKGROUND OF THE INVENTION

The existence of two systems of vasoactive polypeptides, a hypertensorand a hypotensor, in mammal organism is quite new. The fundamental basesfor understanding the hypertensor system, renin-angiotensin system wereestablished through papers of Houssay and Fasciolo (1937), Houssay andTaquini (1938), Braun-Menendez, Fasciolo, Leloir and Mufioz (1939) andKohlstaedt, Helmer and Page (1938). On the other hand, the hypotensorsystem, kallikrein-kinin system are based on Frey, Kraut and Werlepapers, carried out in the 1930 decade (Frey, Kraut and Schultz, 1930;Kraut et al, 1930; Werle, 1936; Werle et al, 1937) as well as Rocha andSilva, Beraldo and Rosenfeld (1949) and Prado, Beraldo and Rocha e Silva(1950).

In the two systems the vasoactive peptide is released to its plasmaticprotein precursor through limited proteolysis according to the followinggeneral scheme:

Several papers on purification and characterization of proteases andsubstrates involved in these two systems allowed the clarification ofseveral steps necessary for releasing the active peptide. However, thephysiological role of the latter, as well as its catabolism is nottotally clarified yet.

The Renin-Angiotensin System

Renin is an acid protease (E.C. 3.4.99.19), produced and stored byjuxtaglomerular cells from afferent arteriole of the renal glomerulus(Kohlstaedt et al, 1938; Hartroft, 1963 and Tobian, 1960). The subtractunder which this enzyme acts is a plasmatic α₂-globulin,angiotensinogen, from which part of the N-terminal sequence is known(Braun-Menendez et al, 1939; Bumpus et al, 1958; Schwyzer and Turrian,1960) which corresponds to:Asp³-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹-Leu¹⁰-Leu¹¹-Val¹²-Tyr¹³-Ser¹⁴.

When renin hydrolisates the Leu¹⁰-Leu¹¹ bond in the angiotensinogenmolecule, decapeptide angiotensin I, which is as not very potentvasoconstrictor, is released. A second enzyme, described by Skeggs et al(1956), called converting enzyme, is the responsible by the hydrolysisof the Phe⁸-His⁹ bond and by releasing octapeptide angiotensin II, whichis pharmacologically active, being inactivated by angiotensinases.

The Kallikrein-Kinin System

The kallikrein-kinin system comprises kininogenases which hydrolisatesan inactive precursor, the kininogen, and releases kinins, which areinactivated by kininases.

The expression kininogenase comprises proteases, such as: kallikreins,trypsin, pepsin, some bacterian proteases and snake poison (Prado, 1970;Rocha e Silva et al, 1949; Suzuki and Iwanaga, 1970). Among theseenzymes, kallikreins are specifics for the system: these areserine-proteases that release kinins of the kininogen, by limitedproteolysis (Neurath, 1975) and have low proteolitic activity on otherproteins. Two types of kallikreins are found in mammals: glandular andplasmatic, which are different each other concerning tophysical-chemical and immunological proprieties, reaction velocity withkininogen and synthetic subtracts, types of kinins released and respondsto a great variety of synthetic and natural inhibitors.

On the other hand, kininogens are acid glycoproteins which contains abradykinin molecule in the C-terminal or next to it (Pierce, 1968); theyare hydrolisated by glandular kallikreins, releasing lysyl-bradykinin,as well as by plasmatic releasing bradykinin (Rocha e Silva, 1974).Lysyl-bradykinin is converted to bradykinin by the existingaminopeptidases contained in plasma (Erdös and Yang, 1970) as well intissues (Hopsu et al, 1966 a, b; Borges et al, 1974; Prado et al, 1975).

Two kininogens functionally different have been described in plasma,namely, a high molecular weight kininogen, which is subtract for the twokallikrein (plasmatic and glandular), and a low molecular weight, whichis a good subtract only for glandular kallikrein (Werle and Trautschold,1963; Prado et al, 1971).

Bradykinin (BK=Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹),lysyl-bradykinin and methionyl-lysyl-bradykinin are strongphysio-pharmaco-pathological agents, which produces hypotension andvasodilation, pain, contraction of the smooth muscle, increases vascularpermeability and leukocyte migration (Erdös and Yang, 1970; Pisano,1975).

Physiological Role of Kinins:

The action of kinins in the organism is not totally clarified, althoughsome attributions have been indicated them as participating in severalphysiological functions, either at systemic or tissular levels.

It is proposed its mediation in different processes such as: periphericvasodilatation and mediation in inflammatory phenomena; interaction withthe synthesis system and prostaglandins release; mobility ofspermatozoids; renal flux regulation; mediation in the sodiumreabsorption by nephron (Wilhelm, 1973; Terragno et al, 1975; Baumgartenet al, 1970; Schill and Haberland, 1974; Levinsky, 1979).

In order to clarify the exact role carried out by kinins in thisprocess, it is important to know not only the mechanism that leads toits release but also to its catabolism.

Catabolism of Kinins:

The responsible enzymes for inactivation of kinins are generically knownas kininases. Under this acronym it is comprised a series of peptidaseswhich are capable to hydrolysate the bonds in BK molecule or itsderivatives, not being necessary or prove to be participant of thekinins catabolism.

Observations on the existence of such class of enzymes have been carriedout since the initial researches of the kinins system and have beendescribed in several organs, tissues and physiological liquids by manyresearchers groups.

Plasma

Two types of kininases have been characterized already in the humanplasma: kininase I (arginine carboxypeptidase, E.C. 3.4.12.7) andkininase II (peptidyl-dipeptidase, E.C. 3.4.15.1).

Kininase I is a carboxypeptidase type enzyme, which was purified for thefirst time from Cohn fraction IV (Erdös e Sloane, 1962). This enzymehydrolysates the Phe⁸-Arg⁹ bonds of BK. It was originally calledcarboxypeptidase-N because of its proprieties, which make them differentof pancreatic carboxypeptidase B. Although its official name, argininecarboxypeptidase, this enzyme catalyses better the lysine C-terminalhydrolysis than arginine, in many substrates (Oshima et al, 1975).

Among synthetic substrates used in the purification and specificitystudies of such enzyme it can be cited the HLA (Oshima et al, 1975).

The second enzyme having kinin activity, described in plasma, is thekininase II, which inactivate BK by hydrolysis of the Pro⁷-Phe⁸ bondsand releases Phe-Arg dipeptides (Yang and Erdös, 1967).

Later, it was observed that such enzyme is identical to the angiotensinI converting enzyme (AI) of the renin-angiotensin system (Yang et al,1970a and Yang et al, 1970b), therefore, being, responsible byhydrolysis of the Phe⁸-His⁹ bond of the AI molecule. One of the featuresof such enzyme is that it is inhibited by potentiator peptides of BK(BPP), described by Ferreira (1965), and Ferreira (1966).

It was also described in other animal species enzymes with similarspecificity to those kininases I e II from human plasma (Erdös and Yang,1970).

Lung

Great importance has been attributed to lung in which concerns BKelimination; many papers published by the literature describes theinactivation by this organ, regarding the high percentage of BK infused(Ferreira and Vane, 1967; Biron, 1968 and Dorer et al, 1974).

The kininase II has been already purified in hog lung (Dorer et al, 1972and Nakajima et al, 1973) rabbit lung and rat lung (Soffer et al, 1974and Lanzillo and Fanburg, 1974).

Studies of Ryan et al have contributed to clarify the mechanism ofinactivation of this organ. BK would be inactivated, while AI would beconverted into AII during the circulation, by an enzyme kininase II typethat was in the pynocitotic vesicles of the vascular endothelium. Ryanet al, also observed that BK is much more easily hydrolyzed than LBK-BKand MLBK. Their theory is that these bigger kinins have a more difficultaccess to the vesicles. According to Ryan et al statement the BKhydrolysis products which were found after the lung circulation would beconsequence not only from the action of the first cited enzyme but alsofrom the action of other enzymes contained in the cytoplasm ofendothelial cells (Ryan et al, 1968 and Ryan et al, 1975).

Liver:

Erdos and Yang attributed almost exclusively to the plasmatic andpulmonary kininases the responsibility for the kinin catabolism “invivo”. Researches carried out by Prado et al (1975) show, however, thatother organs are able to inactivate kinins when they are perfused inweak rats, in which lung circulation was excluded from the perfusioncircuit. In the referred paper when liver is perfused “in situ”, it wasshown that the organ inactivates considerable quantity of BK.

Following these researches, Borges et al (1976) observed that BKinactivation by the perfused liver “in situ” is due to, at least, twoenzymes: a peptidyl dipeptide hydrolase and a second one that hydrolyzesthe Phe⁵-Ser⁶ bond of BK. This enzyme could be a membrane peptidase,since it was removed from the perfused liver through the use of TritonX-100 in the perfusion liquid. According to the authors, the kininactivity obtained in this research is, very low when compared to thosefound in the supernatant of the total homogenate of the organ.

Mazzacoratti (1978) have worked with a preparation of this type, thatis, homogenized liver from rats. It was purified two serine-proteaseshaving different molecular weight which hydrolisates the Phe⁵-Ser⁶ bondof BK.

Brain

It has been studied by many researchers the metabolism of kinins inbrain extracts (Iwata et al, 1969; Camargo et al, 1969).

Kininases from homogenized rabbit brain have been systematically studiedby Camargo et al (1973), Oliveira et al (1976). Two thiol-endopeptidasesoptimum pH 7.5 were purified from the supernatant fraction. The firstenzyme, kininase A, hydrolyzes the Phe⁵-Ser⁶ BK bond and has a molecularweight of 71 kDa; while the other, kininase B, hydrolysates Pro⁷-Phe⁸ askininase II, but it has a molecular weight of 6900. This enzyme would bedifferent from the converting enzyme (kininase II), since preliminarystudies did not show the conversion of AI into AII.

Wilk, Pierce and Orlowiski (1979) described two enzymes from braintissue which differs from the referred above. One of the enzymes, whichwas extracted from the bovine pituitary, also hydrolysates the Phe⁵-Ser⁶Bk bond, however because of its molecular weight (higher than 100000)and because it is inhibited by Na⁺ and K⁺ it differs from kininase A.The second enzyme described, which was extracted from rabbit brain, isspecifically for hydrolysis of those peptide bonds in which prolinecontributes with carboxyl group. This enzyme firstly hydrolyses thePro⁷-Phe⁸ BK bond and secondly, the Pro³-Gly⁴ bond.

Kidney:

The kininase activity of kidney is higher than found in plasma or liver(Erdös and Yang, 1970).

Several enzymes have been purified, in this organ, with kininaseactivities. Researches of Erdos et al, have identified three differentenzymes in the kidney: one, carboxypeptidase type, which releasesarginine C-terminal of BK, which differs from some properties ofplasmatic kininase I, this is why it was called kininase p (Erdös andYang, 1966); another enzyme, which hydrolisates the Pro⁷-Phe⁸ bond(Erdös and Yang, 1967), and a third one, characterized as animidopeptidase, which inactivates BK by hydrolysis of the Arg¹-Pro² bond(Erdös e Yang, 1966).

Koida and Walter (1976) purified, from sheep kidney, an enzyme thathydrolysates Pro-x bonds type in the molecule of several peptides, amongwhich the BK. It was observed that the x aminoacid cannot be proline andthat its catalysis is faster if x is a lipophilic aminoacid.

The kinin catabolism by the kidney has been studied by methods aiming atto identify the inactivation sites of such peptides. These studiesindicate that, besides the BK hydrolysis that occurs at vascular networklevel, the catabolism of kinins by enzymes located at renal corticalcells seems to have great importance (Erdös and Yang, 1967).

The kininase activity is very low in the glomerulus, but a type IIkininase is found in great concentration in brush border of the proximalconvoluted tubule (Holl et al, 1976, Casarini et al, 1997). In agreementwith this discovery, Oparil et al (1976) observed that a high percentageof BK microinfused is inactivated in the proximal tubule. Consideringthat the kinin generation in kidneys should occur close to the distaltubule, where kallikrein is synthesized (Ørstavik et al, 1976), it seemsto be logical to suppose that from this point, other kininases should bepresent and the nephron or in the intratubular fluid.

Urine

A carboxypeptidase was well characterized by Erdös et al (1978), inhuman urine; it releases BK C-terminal arginine and differs from theplasmatic one as to molecular weight, inhibitors action andimmunological proprieties. However, kinetic and inhibition similaritiesto renal enzyme are shown.

Ryan et al described, in 1978, three enzymes contained in urine: oneenzyme hydrolyzates the Pro⁷-Phe⁸ BK bond and transform AI into AII;another enzyme, having 63 kDa molecular weight, which breaks thePhe⁸-Arg⁹ BK bond, is not inhibited by BPP_(9a).

Figueiredo et al (1978) also described, a kininase having molecularweight of 250 kDa, which is inhibited by chelate agents that would besimilar to the third among those described by Ryan. This enzymehydrolysates C-terminal arginine, although it does nor hydrolysates theHLA synthetic substrate.

With the exception of Erdös et al, 1978, research that have purified andcharacterized a carboxypeptidase from urine, all researches, however,the described enzymes were only partially purified and/or characterized.Due to these contradictory data, the present invention aims at tocharacterize the different kininase activities that are the ACE in humanurine.

One of the forms of low molecular weight (LMW) of the angiotensin Iconverting enzyme of 91 kDa was observed during the preparation of suchenzyme from rat lung homogenate [Lanzillo et al, 1977]. This LMW formfrom ACE was also observed in human lung [Nishimura et al, 1978], hogkidney [Nagamatsu et al 1980] and human kidney [Takada et al, 1981].Iwata et al (1983) and Yotsumoto et al (1983) have shown that ACE LMW of86-90 kDa can be obtained from rabbit lung and human plasma,respectively, after treatment with bases. In the 90′ Lantz et al (1991),described three different ACE isoforms having molecular weights of 150kDa, 80 kDa and 40 kDa characterized in the human cerebrospinal fluid.All the previously referred are similar to somatics. Casarini et al,1991, 1995, 2001, described the 65 kDa and 90 kDa isoforms, bothN-domain in hypertense patients urine and 65 kDa on normal persons.Deddish et al (1994) purified an ECA with 108 kDa molecular weight inileal fluid, which is also an N-domain isoform of ACE.

It has been described the purification of several isoforms of ACE [Ryanet al, 1978; Kokubo et al, 1978; Skidgel et al, 1987; Casarini et al,1983, 1987]. Kokubo et al (1978) found three different forms of ACEnormal human urine. Two forms with high molecular weight of >400 kDa and290 kDa and a third one, molecular weight of 140 kDa. Ryan et al (1978)described a kininase II human urine that was separated in two forms. Thefirst co-cromatography with somatic ACE of 170 kDa, and the second wassimilar to a protein having molecular weight of 90 kDa. Casarini et al(1983, 1991, 1992, 1995, 2001) described he ACE in human urine of normalpersons and hypertense patients with molecular weights of 190 kDa, 90kDa and 65 kDa and also in rat urine (Casarini et al 1987). Alves et al,1992 also described isoforms of 170 kDa, 90 kDa and 65 kDa in urine ofnormal persons and hypertense patients. Costa et al, 1993, 2000described in normal persons urine, ACE with different molecular weightsof 170 kDa, 65 kDa and 59 kDa, and in the hypertense patients,renovasculares enzymes with molecular mass of 55 kDa, 57 kDa e 94 kDa.The ACE activity in urine is not from the plasma but from the renaltubule (Casarini et al, 1997) and can be used as a reference for therenal tubular damage, since there is a considerable level increasing inrenal and infections of upper urinary treat diseases [Baggio et al,1981; Kato et al, 1982].

It was also recently described, two ACE isoforms in intracellular andextracellular medium of mesangial cells in culture, having molecularweight of 130 kDa and 65 kDa (Andrade et al, 1998). It was stillobserved the presence of 190 kDa and 65 kDa isoforms in children urinebut in premature children only 65 kDa isoforms ACE, being the lattersimilar to the N-domain portion of the same. In premature children, itwas found, in a period of 1 to 30 days after they were born, that these190 kDa isoform would appear only in the thirtieth day (Hattori et al,2000).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (A, B, C): Shows a gel Chromatography filtration in Ac A-34column of human urine.

FIG. 1A: Normotensive children/normotensive parents (At 280 nm—ACEActivity on the HHL substrate).

FIG. 1B: Normotensive children/hypertensive parents (At 280 nm—ACEAtivity on the HHL substrate).

FIG. 1C: Hypertensive children/hypertensive parents (At 280 nm—ACEAtivity on the HHL substrate).

Urine of normotensive persons with hypertensive parents has presentedthe three ACE isoforms having 190 kDa, 90 kDa e 65 kDa molecularweights, showing that the 90 kDa premature isoform appears. Thus,showing to be a prognostic that these persons (individuals) could gethypertension, being, therefore, a genetic marker for hypertension.

FIGS. 2A and 2B: Presentation of N-terminal and C-terminal sequence of90 kDa and 65 kDa angiotensin I isoforms converting enzymes. The 65 kDaenzyme ends at the number 481 aminoacid. The 90 kDa enzyme ends atnumber 632 aminoacid.

FIG. 3: Fresh human urine Western Blotting—Line 1: normal person urine,Line 2: wild ACE recombinant, Line 3: ACE recombinant secreted, Line 4:hypertensive patient urine.

FIG. 4: Scheme of dosage by mass spectrometer—the scheme presents 5(five) steps which starts by raw urine that in the second step iscentrifuged for 10 minutes at 3000 rpm speed and at 4° C. temperature.In the third step, urine is concentrated 4× (four times) in a Ultrafree(Millipore) tube and is centrifuged during 5 (five) minutes at 3000 rpmand 4° C., then going to a forth step where concentrated urine passthrough a dialysis in centricon Tris/HCL 1 mM buffer, pH 8.0 centrifugedduring 5 (five) minutes at 3000 rpm and 4° C., resulting in aconcentrated urine and dialyzed and finally, the fifth step whichresults the HPLC-MS.

DESCRIPTION OF THE INVENTION

In order to carry out the method of identification and quantification ofproteins, isoforms of the angiotensin I converting enzyme, specifically190-kDa ACEs, specially 90 kDa and 65 kDa in tissues, cells andbiological fluids, specially in urine, according to the presentinvention, it starts with collecting fluids, such as urine, tissues orcells from living organisms, submit them to a chromatographic separation(AcA44 and/or AcA 34 resin; reverse phase column C-18, in massspectrometer) and by Western Blotting (using a specific antibody againstsomatic ACE and N-domain ACE [90 kDa, genetic marker for hypertensionand 65 kDa] of 190 kDa, 90 kDa and 65 kDa isoforms. Normal individualshave the 190 kDa and 65 kDa isoforms while the 90 kDa isoform(hypertension genetic marker) will characterize those individualpredisposed to develop hypertension and lesion in characteristic targetorgans (heart, nervous system, vascular system and kidney).

The method of the present invention considers that an aliquot of fluid(for example, fresh or concentrated urine), cells and tissues areprocessed and analyzed by high performance liquid chromatography anddetection by mass spectrometry (HPLC-MS) or directly in the massdetector, where the sample is analyzed and compared with the previouslyestablished standards for 190 kDa, 90 kDa (hypertension genetic marker)and 65 kDa ACE isoforms. An aliquot of fluid (for example, fresh orconcentrated urine), cells and tissue are processed and analyzed byWestern Blotting or another immunoprecipitation method) using specificantibodies against 190 kDa and N-domain ACE (90 kDa hypertension geneticmarker and 65 kDa), using as a control analysis the ACE isoformsprepared as standards as well as the ECA recombinant enzyme.

In order to reach the results proposed by the present invention, theresearches on ACE, started in 1983, when essential (light and/or mildforms) arterial hypertensive patients were analyzed after usingcaptopril (50 to 150 mg) orally administered in one-day dose. Three daysstudy, each day, each six hours collect duration; samples of blood andurine were collected at the final basal period (1 hour), after theyreach the supine position, as well as at the end of the study (after 6hours). The results show a 50% inhibition on the enzyme activity, in aperiod between 1 and 2 hours after captopril administration, returningto the basal levels at the end of the studies. The enzymatic activityusing Hipuril-His-Leu substrate was measured by Friedland andSilverstein method (1976). Through ion exchange chromatography analysis,the collected urine (collected after 6 hour) two protein peaks wereeluted with angiotensin I converting activity and inactivated forbradykinin , in conductivity of 0.7 mS (90 kDa) and 1.25 mS (65 kDa),differing from the profile found in 190 kDa e 65 kDa ACE in normotensiveindividuals. Based on this, studies have been developed with a highernumber of light hypertensive patients, where it was found the presenceof two protein peaks with angiotensin I converting activity, as citedabove (Casarini et al, 1991).

Following the studies as referred above (project support by FAPESP, No95/9168-1) the following study groups were organized: normalparents/normal children; normal parents/hypertensive children;hypertensive parents/hypertensive children and hypertensiveparents/normal children. It was found that the group (normalparents/hypertensive children) hypertensive parents/hypertensivechildren, the children urine was presented two 90 e 65 kDa forms; in thenormal parents/normal children group, the children urine presented the190 and 65 kDa forms; and, finally, in the hypertensive parents/normalchildren, the children urine presented the 190, 90 and 65 kDa forms,being these two last forms, N-domain fragments.

From these results, it was concluded that 90 kDa would possibly be ahypertension marker. In order to prove if this findings was a geneticfactor or another fact related or not to pressure increasing (physical),data were validated in the same project in the experimental model forrats. For this purpose, it was studied Wistar Brown Norway, Lyon, SHR,1R1C and DOCA-salt rats urine. As a result, the Wistar, DOCA-salt, 1R1Cand Brown Norway rats presented 170 and 65 kDa forms; only SHR andSHR-SP rats presented the 90 and 65 kDa forms. These results confirmthose obtained for humans, thus, being, the 90 kDa ACE isoform ahypertensive genetic marker (Fapesp 97/00198-0, Marques 1999).

In 1997, researchers of the present invention described that themesangial cells in a culture that expresses the ACE RNAm (Casarini etal, 1997) . This enzyme is detected in the intracellular (136 kDa and 65kDa) and secreted (136 kDa e 65 kDa), indicating a potential effect ofthe local production of angiotensin II in the function of these cells(Fapesp 95/9168-1; Andrade et al, 1998).

It was observed latter that the intracellular and the culture means ofthe SHR mesangial cells presented the same profile of (90 and 65 kDa)ACE isoforms, which were found in urine of such rats (Fapesp99/01531-1); therefore, this confirms the results of the previousresearches. From these results, it was observed by the authors of thepresent invention, that these isoforms are expressed in the lung,adrenal, heart, aorta and liver of Wistar rats (136 kDa and 69 kDa) andSHR (96 kDa and 69 kDa) and are not restricted to kidney (Ronchi, 2002);emphasizing that the 80/90/96 kDa hypertension genetic marker isexpressed in the various tissues and, therefore, bringing to concludethat these isoforms can contribute for a regulation of the specificorgan (it should be stressed that when de 80 or 96 or 90 kDa enzymes arereferred, it should be understood, the same enzymes with smallalteration in the glycolization process).

The present invention starts with fluid collect as for example urine,tissue or living organisms cells, that are submitted to achromatographic separation (resin AcA44 and/or AcA 34; phase reversecolumn C-18, mass spectrometer) and by Western Blotting (using aspecific antibody against ACE somatic and against ACE N-domain [90 kDa,hypertension genetic marker and 65 kDa] of 190 kDa, 90 kDa and 65 kDaisoforms. The 190 kDa and 65 kDa isoforms will be present normalindividuals (normal rats, cells and/or tissue from normal rats); whilethe 90 kDa (hypertension genetic marker) isoform will characterize those(individuals or animals, etc) predisposed to develop hypertension andlesions in characteristics target organs (heart, nervous system,vascular system and kidney). A aliquot of fluid (for example, fresh orconcentrated urine), cells and tissue are processed and analyzed by highperformance liquid chromatography method and detection by using massspectrometry (HPLC-MS) or directly in the mass detector, where thesample is compared with the standards established for ACE of isoforms190 kDa, 90 kDa (hypertension genetic markers) and 65 kDa. An aliquot offluid (for example, fresh or concentrated urine), cells and tissue areprocessed and analyzed by Western Blotting using specific antibodiesagainst 190 kDa ACE and N-domain ACEs (90 kDa), hypertension geneticmarker and 65 kDa), using as analysis control the ACE isoforms preparedas standards and the recombinant ACE enzyme.

Ace Isoform as Hypertension Marker

Isoform of Angiotensin I Converting Enzyme (90 kDa, N-Domain) as aHypertension Genetic Marker Produced by Human Urine:

Based on the previous studies, the researchers of the present inventionfound in normotensive individuals urine and using ion exchangechromatography, two peaks of angiotensin I converting activity withmolecular mass of 196 kDa and 65 kDa. When hypertension patients urineis processed, it was obtained a profile where two peaks were eluted withangiostensin I converting activity in the 90 kDa and of 65 kDa molecularweight, not being detected the 190 kDa form (Hypertension 26:1145-1148,1995).

One of the objectives of the present invention consists in confirmingthe potential of the de 90 kDa isoform as a hypertension genetic markerand as a hypertension prognostic.

The following study groups were established for this purpose:

-   -   normotensive individuals with normotensive parents,    -   normotensive with hypertensive parents    -   hypertensive with normotensive parents, and    -   hypertensive with hypertensive parents.

The collected urines were concentrated separately and dialyzed withTris-HCl 50 mM buffer, pH 8.0 and then submitted gel filtration inAcA-34 column equilibrated with Tris-HCl 50 mM buffer, containing NaCl150 mM, pH 8.0. The collected fraction (2 mL) have been monitored byreading the absorbance in 280 nm and by the angiotensin I convertingactivity, using Hipuril-L-His-L-Leu- and Z-Phe-His-Leu as substrates.The following results was obtained: normotensive individuals withnormotensive parents presented two isoforms with ACE activity (190 kDaand 65 kDa) (n=21); normotensive individuals with hypertensive parentspresented three (190 kDa, 90 kDa and 65 kDa) (n=13) isoforms andhypertensive individuals with hypertensive parents presented twoisoforms (90 kDa and 65 kDa) (n=13). As expected, it was not foundanybody that would constitute the hypertensive group with normotensiveparents.

Two individuals that presented 190 kDa, 90 kDa and 65 kDa isoforms,normal pressure, and that were in contact with the research group, weremonitored for 4 years. In the forth year after detection of isoforms inthe urine, they became hypertensive; this proves, therefore, that the 90kDa isoform is really a hypertensive genetic marker.

Conclusion:

Considering that the urine of normotensive individuals with hypertensiveparents presented the three ACE isoforms with molecular weight of 190kDa, 90 kDa and 65 kDa, shows that the 90 kDa isoform which earlyappears, is a prognostic that these individuals could be a hypertensiveperson, thus, being, a hypertensive genetic marker.

Quantification and Identification of the Ace Isoform, HypertensionGenetic Marker by Mass Espectrometry and Western Blotting

Western Blotting of the Human Fresh Urine:

Urine was collected from a single time in the presence of a “pool”(several inhibitors) of proteases, then, it was concentrated and 100 ugwas submitted to a 7.5% polyacrylamide gel electrophoresis, followed byWestern Blotting with PVDF membrane, then it was incubated with thepolyclonal antibody Y1 against human ACE. Line 1: urine of normalindividual, Line 2: ACE wild recombinant, Line 3: ACE recombinantsecreted, Line 4: hypertensive individual urine.

Dosage for Mass Espectrometer:

Raw urine was centrifuged for 10 minutes, at 3000 rpm, 4° C., followedby 4× concentration in Ultrafree (Millipore) tube, then centrifuged for5 minutes at 3000 rpm, 4° C., dialyzed with centricon Tris/HCl 1 mMbuffer, pH 8.0. Then, it is centrifuged for 5 minutes, at 3000 rpm, 4°C. Concentrated and dialyzed urine was, then, obtained and therefore,the prepared sample was analyzed in HPLC-MS.

The solvents used in the HPLC system were: solvent A, which consists of0.1% trifluoroacetic acid (TFA, Merck, Germany). The urines wereseparated in a Nova Pak C¹⁸ (Waters) reverse phase column, for 15minutes with a 1.5 mL/min flux. The conditions are still beenstandardized in order to improve the method resolution.

Isoform of Angiotensin I Converting Enzyme (90 kDa) as a HypertenssiveGenetic Marker Secreted in Rats Urine. Protocol Design to Prove theFindings (Affirmation of Genetic Marker for the 90 kDa Protein) withHuman Urine.

ECA isoforms presented in isogenic normotensive rats (WKY and BrownNorway) urine have been identified as well as in normotensive, isogenichypertensive (SHR, SHR-SP, Lyon), isogenic normotensive, experimentallyhypertensive (1K1C and DOCA-Salt) and isogenic hypertensive rats, whichwere treated with antihypertensives drugs (SHR+enalapril), aiming at tocompare the obtained chromatography profiles, and with the objective tocharacterize the 90 kDa form as an arterial hypertensive genetic marker.

From WKY rats urine, two peaks of AI converting activity have beenseparated by gel filtration in AcA-44 resin: the first, WK-1,corresponds to a high molecular enzyme (190 kDa) and the second one,WK-2, corresponds to a low molecular weight (65 kDa); these data wereconfirmed by Western Blotting. In the SRH group, the chromatographicprofile have presented different results from the previous group (WK),being identified an ACE called S-1, with molecular weight 80 kDa, and asecond one, S-2, molecular weight of 65 kDa, similar to those found inhypertensive patients urine that was not 90 kDa and 65 kDa treated(Casarini et al., 1991). The molecular mass differences between the 80kDa enzyme from rat and the 90 kDa enzyme of human urine occur due tothe glycolization process (data not shown).

In the third group (1K1C), a renovascular induced hypertension model,the chromatography profile was similar to the one found in rats used ascontrol (WKY). In this group two peaks of AI converting activity wereobtained: the first, C-1, corresponds to a 190 kDa enzyme and the secondone, C-2, to 65 kDa.

Two peaks of AI converting activity were separated by gel filtration inAcA-44 resin, from SHR-SP rats urine: the first one, called SP-1,corresponds to a 80 kDa enzyme and a second one, called SP-2, whichcorresponds to the 65 kDa enzyme, similar to that found in SHR ratsurine and also found in not-treated hypertensive patients urine(Casarini et al, 1991, 1995).

On the other hand, the SHR rats, which were treated with enalapril, showthat although having their pressure under control, they carry the 80 kDaisoform; this fact shows that the isoform profile is linked to geneticfactors.

In the group of rats used as control, the DOCA-Salt model, in which itwas not administered the hypertensive treatment, the chromatographicprofile was similar to that found for normotensive WK rats. Two peakswith AI converting activity were obtained: the first, CD-1, correspondsto a 190 kDa enzyme, and the second, CD-2, corresponds to the 65 kDa.

The DOCA-Salt model, with reduced hypertension induced by DOCA andsaline administration, presented a chromatographic profile similar tothat found in DOCA-Salt and WK control rats. Two peaks of AI convertingactivity were obtained: the first one, D-1, corresponds to 190 kDa ECA,and the second one, D-2, to 65 kDa ECA. This result shows that the 80kDa presence is linked to a genetic factor not being consequence of theincreasing of pressure.

The result of the gel filtration in Brown Norway normotensive rats urinewas similar to the profiles found in normotensive rats urine. Two peakswith ACE activity have been obtained: BN-1, which corresponds to the 190kDa enzyme, and the second one, BN-2, which corresponds to the 65 kDaenzyme, showing that different strain of normotensive presents the samechromatographic profile.

Comparing the chromatographic profiles of WK rats (normal control)urine, 1K1C (experimental hypertensive—Goldblatt), DOCA-Salt (control),DOCA-Salt (experimental hypertensive) and normotensive Brown Norway withthe urine of SHR rats, Lyon and SHR-SP (genetically hypertensive), itcan be affirmed that the basic difference is the presence de 80 kDaisoforms in the genetically hypertensive rats urine. The fact that the80 kDa isoform do nor appear in the 1K1C and DOCA-Salt rats, whosehypertension is induced (physical factor), reinforces the hypothesisthat the same is linked to a genetic factor.

Conclusion:

The results found suggest that rats, genetically predisposed tohypertension, the 80 kDa form would be detected instead of 190 kDa; thiswould be used to, as a consequence, as an early genetic marker forhypertension.

Segregation of the Isoform of Angiotensin I Converting Enzyme (90 kDa,N-Domain), Hypertension Genetic Marker in Rat Urine. Protocol Design toShow the Presence (Segregation of the Hypertension Genetic Marker(Protein of 90 kDa) in Rats Urine. Rats Crossing ExpontaneouslyHypertenses (SHR) and Brown Norway (BN).

In a previous project it was characterized the different isoforms of lowmolecular weight in rats urine in different experimental models(Wistar-Kyoto, SHR, 1R1C, DOCA-salt control, DOCA-salt, SHR-SP and BrownNorway). It was observed that the 90 kDa form only appears in SHR andSHR-SP rats, showing a genetic factor for the presence of such a form.In this project, it is studied the isoforms gene transmission of 190kDa, 80 kDa and 65 kDa of the angiotensin I converting enzyme, genotypeand phenotype analysis in rats urine generated from the crossings andbackcrossings among SHR and Brown Norway races.

Drawing of the Crossing

Crossings were carried out between Brown Norway and SHR (BN×SHR), ratsgenerating a group of heterozygotes rats called F1SB01 to F1SB04; fromthis group two animals were chosen (F1SB01 e F1SB03), males in order tocarry out the backcrossing with the SHR rat (female). For phenotyping,the urine of the animals was collected and concentrated, then, it wassubmitted to a AcA-34 gel filtration column chromatography, togetherwith Tris/HCl 0.05M buffer, pH 8.0, containing NaCl 0.15M. Fractions of2.0 ml have been eluted under a 20 ml/h flux, being monitored byabsorbance measured in A280 nm and by the ACE enzymatic activity usingZ-Phe-His-Leu (ZPheHL) as a substrate.

Results

Parents: two peaks with ACE activity were eluted from BN rats urine andsubmitted to AcA-44, BN-1 e BN-2 column chromatography, with molecularweight estimated of 190 kDa and 65 kDa, respectively. On the other hand,in SHR rats urine it was found two peaks with converting activity,however, with molecular weight estimated of 90 kDa and 65 kDa,respectively.

In F1, 39 animals were generated, from which 100% were phenotyped asheterozygotes for the three, 190, 90 and 65 kDa enzyme forms. From thebackcrossing animals were generated from which 85% present the threeenzyme isoforms (NH group) and 15% presented the 90 and 65 kDa forms (Hgroup).

Conclusion

Through the obtained results, it is suggested that the 90 kDa isoforms(arterial hypertension genetic marker) continue to be present in thegenerations originated by the crossings and backcrossings.

Expression of the Hypertension Genetic Marker, 90 kDa Isoforms inTissues (Aorta, Adrenal, Heart, Liver, Lung, Kidney, Pancreas) of RatsExpontaneouly Hypeertensives Compared With to the Wistar and IsogenicWistar.

It as been identified in previous studies 190 and 65 kDa in Wistar,whose profile, similar to those described for normotensive individuals.80 and 65 kDa isoforms have been identified in SHR rats urine whileN-terminal ECA fragments repeats the profile, which was found for lighthypertensive individuals.

The homogenates were submitted to a gel filtration chromatography andtwo peaks have been detected, having activity on the HHL substrate indifferent W and W1 rats tissue, whose molecular weights of 137 and 69kDa are similar to those referred for W rats (Table I). The SHR ratstissues also presents two peaks of activity whose estimate molecularweights are 96 e 69 kDa, and this profile corresponded to the one foundfor the enzymes contained in the urine of said rats. The proteinexpression of the 137 and 69 kDa isoforms was observed in all tissues,which have been studied, obtained from Wistar and isogenic Wistar ratsthrough the Western Blotting technique. By using the same technique, 96and 69 kDa isoforms expression of all tissues of the SHR rats have beenconfirmed (Table I). The obtained results show the expression of the 69kDa isoforms (besides the 137 kDa isoforms) in the W and WI tissues, asthe 96 and 69 kDa isoforms in SHR rats tissues, bringing to theconclusion that the expression of N-domain isoforms, more specificallythe 96 kDa isoform (hypertensive genetic marker) is not restricted onlyto urine and/or kidney, but are also present, locally, in the studiedtissues.

Table I—Sumary of the Study as to Elution Fractions and EstimatedMolecular Masses, Showing the 80 kDa ECA as Hypertension Genetic Marker.Estimated Elution Molecular Weight Strains Enzymes Fraction (N°) (kDa)WKY WK-1 32 190 WK-2 54 65 SHR S-1 50 80 S-2 55 65 1K1C C-1 32 140 C-254 65 DOCA-Salt D-1 34 190 D-2 52 65 DOCA-Salt CD-1 34 190 Control CD-252 65 SHR-SP SP-1 50 90 SP-2 55 65 BN BN-1 32 190 BN-2 54 65 SHR S-1 5080 enalapril S-2 55 65 Lyon L-1 50 80 L-2 55 65Expression of the Hypertension Genetic Marker, 90 kDa Isoforms in theMesangial Cells in Rats Culture, Expontaneously Hypertensesives Comparedto the Wistar Rats

The glomerulus has been isolated from Wistar or SHR rats, according toGreenspon and Krakomer method (1950). The rats were put under sulfuricether atmosphere and submitted to a bilateral nefrectomy. Kidneys weredecapsulated and cortical macrodissecation was carried out. The cortexwas separated from the medula and then, fragments were passed throughseries of sieves which differ in size according to the meshes openings(60, 100 and 200 mesh). The glomerulus were collected from the surfaceof the third sieve and forced to pass through a (25×7) needle (aiming atto decapsulate the glomerulus. The decapsulated glomerulus were countedby using Newbauer chamber and divided (density of ˜300 glomerulus/cm²)in 25 cm² bottles, using RPMI 1640 supplemented with 20% of bovine fetalserum, penicillin (50 U/mL), HEPES (2.6 g) and glutamin 2 mM. Theculture bottles were maintained into CO₂ (5%), at 37° C. Each 36 h themedium was changed. After 3 weeks approximately the primary culture ofthe mesangial cells were submitted to trypsin. The subcultures grew inthe same medium. This procedure was repeated up to the thirdsubcultured, when cells were prepared for the experiments: Mesangialcells (MC) (3° subcultured) were incubated for 20 hours with RPMI,without bovine fetal serum; further, the MC and the medium was collectedseparately.

The collected mean in the 3° subcultured was concentrated in an Amiconconcentrator. The concentrated medium (2.0 mL) was submitted to a gelfiltration in AcA-44 (1.5×100.8 cm column; volume 178.0 mL),equilibrated with Tris-HCl 50 mM buffer, pH 8.0, containing NaCl 150 mM.Fractions of 2.0 mL under flux of 20 mL/h were collected. Elution wascarried out under a 20 mL flux by one hour. Fractions of 2 mL werecollected, and monitored by absorbance measurements in 280 nm and theenzymatic activity was quantified, by using Hippuryl-His-Leu (HHL).

The CM collected were lysed with 4 mL Tris-HCl 50 mM buffer, pH 8.0,containing Triton X-114 1% and PMFS 0.5 mM, through mechanicalagitation, by one hour, at 4° C. After this period, the lysed cells werecentrifuged and the supernatant was collected and concentrated an Amiconconcentrator, under nitrogen pressure, at 3 kgf/cm². Further, 2mL wassubmitted to a gel filtration chromatography.

The results obtained for MC in Wistar and SHR rats culture presented thesame chromatographic profile obtained for human urine of normalindividuals and moderated hypertension patients as well as for urine ofWistar and SHR rats, thus, confirming that in kidneys, more specificallyin the glomerulus, the different isoforms, already mentioned areproduced (Table II). TABLE II Summary of the study groups as todeterminate molecular masses. Tissues Wistar Wistar Isogenic SHR Adrenal137 137 96 69 69 69 Aorta 137 137 96 69 69 69 Heart 137 137 96 69 69 69Liver 137 137 96 69 69 69 Lung 137 137 96 69 69 69 Kidney 137 137 96 6969 69 Testicle 137 137 96 69 69 69Final Conclusion:

Based on the fact that 90 kDa ACE only appears in hypertensivepatients/MC of SHR/urine of SHR rats, it is suggested that it could havean important and specific role as a hypertension genetic marker.

Based on the studies obtained with the parents and children there wasobserved that 190, 90 e 65 kDa isoforms are present in normalindividuals from hypertensive parents showing that a segregation of thisisoform, thus, it could be characterized as a hypertensive predictor.These data were confirmed in crossing and backcrossing of Brown Norwayand SHR rats (Table III). TABLE III ECA isoforms detected in theextracellular and lysed cells mesangial cells in Wistar and SHR ratsculture. Rats Extracelular Intracelular Wistar 130 kDa  135 kDa  60 kDa68 kDa SHR 80 kDa 80 kDa 60 kDa 68 kDa

References

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1. A method of identification and quantification of proteins, isoformsof angiotensin I converting (ACE) in tissues, cells and biologicalfluids comprising the following steps of: (a) collecting an aliquot offresh or concentrated biological fluids, cells or tissues of livingorganisms and submit them to analysis and separation by Western Blottingmethod; (b) comparing the sample under analysis to the previousestablished standards for the hypertensive genetic markers and 65 kDa,isoforms of ACE 190 kDa, 90 kDa and 65 kDa, an aliquot of fluid (forexample, fresh or concentrated urine) using, as analysis control, ACEisoforms prepared as standards and the ACE recombinant enzyme; and (c)detecting the 190 kDa and 65 kDa isoforms in normal individuals and alsodetecting the presence of 90 kDa isoforms that is going to characterizethose predisposed persons for developing hypertension and lesions incharacteristic target organs.
 2. A method according to claim 1, whereinthe 90 kDa isoform, which was detected in step (c), is a hypertensiongenetic marker and a prognostic agent for hypertension.
 3. A methodaccording to claim 1, wherein said separation of step (a) is processedAcA44 and/or AcA 34 resin, C-18 reverse phase column C-18, massspectrometer, and Western Blotting using a specific antibody againstsomatic ACE and against N-domain ACE [90 kDa and 65 kDa] of 190 kDa, 90kDa and 65 kDa isoforms.
 4. A method according to claim 1, wherein thebiological fluid is urine.
 5. A method according to claim 2 wherein itis detected in urine of normotensive individuals, two peaks withangiotensin I converting activity with 190 kDa and 65 kDa molecularweights.
 6. A method according to claim 5, wherein ion exchangechromatography is used.
 7. A method according to claim 4 wherein it isdetected in hypertensive individual urine a profile where it was elutedtwo peaks with angiotensin I converting activity with 90 kDa and 65 kDamolecular weights, not being detected the 170 kDa form.
 8. A method ofidentification of the potential of 90 KDA isoform of angiotensin Iconverting enzyme comprising the following steps: (a) concentrating anddialyzing dialyzed urine with Tris-HCl 50 mM buffer, pH 8.0 and submitit to a gel filtration in AcA-34 column equilibrated with Tris-HCl 50 mMbuffer, containing NaCl 150 mM, pH 8.0; (b) collecting 2 mL from thefractions and monitoring them through absorbance measurements at A280 nmand by the converting activity of angiotensin I, usingHipuril-L-His-L-Leu-and Z-Phe-His-Leu as subtracts; and (c) observingthe presence of isoforms with ACE activity (170 kDa and 65 kDa) (n=21),from isoforms (170 kDa, 90 kDa and 65 kDa) with (n=13) as well as (90kDa and 65 kDa) (n=13) isoforms.
 9. A method according to claim 8,wherein the two isoforms with ACE activity (170 kDa and 65 kDa) (n=21)detected in the step (c) are from normotensive individuals withnormotensive parents.
 10. A method according to claim 8, wherein thethree isoforms (170 kDa, 90 kDa and 65 kDa) (n=13) detected in step (c)come from normotensive individuals with hypertensive parents.
 11. Amethod according to claim 8, wherein the two isoforms (90 kDa and 65kDa) (n=13) detected in step (c) come from hypertensive individuals withhypertensive parents.
 12. A method according to claim 8, wherein the 90kDa isoform is a hypertension genetic marker and a prognostic agent forhypertension.
 13. A hypertension genetic molecular marker based on saidgenetic proteins obtained according to claim 1, wherein it is the basisof the 90 kDa isoform.
 14. Use of genetic marker obtained according toclaim 1, wherein it is used as a prognostic agent of hypertension. 15.Use of genetic marker obtained according to claim 1, wherein it is usedin the diagnosis of the predisposition for the development ofhypertension and lesions in characteristic target organs.
 16. Useaccording to claim 15, wherein the target organs are the heart, nervoussystem, vascular system and kidney.
 17. An analytical method fordiagnosis, risk stratification, therapeutical decision in carriers ofarterial hypertension and renal lesions wherein the presence of the 190kDa and 65 kDa isoforms are detected in normal individuals, it isdetected the 90 kDa isoform presence that is going to characterize thosepredisposed individuals to the
 18. A method according to claim 17,wherein the 90 kDa isoform is a genetic marker of hypertension and aprognostic agent of hypertension.
 19. A method according to claim 17,wherein the biological fluid is urine.
 20. A kit for diagnosis furthercomprising the genetic marker obtained according to claim
 1. 21. A kitfor diagnosis further comprising a genetic marker and a prognostic agentof hypertension.
 22. A kit according to claim 16, wherein said kit isused in diagnosis, risk stratification and therapeutical decision in thearterial hypertension.